Method of treating hyperlipidemia and atherosclerosis with mir-30c

ABSTRACT

This disclosure provides a novel role for microRNA (miR) regulation of lipid metabolism via the MTP pathway, leading to reductions in apoB secretion and blood lipid levels. MiR regulation of the MTP pathway is shown herein to reduce hyperlipidemia and atherosclerosis in vivo. Therefore, inhibition of MTP expression and activity by miR regulation is identified as a new therapeutic target for treatment of cardiovascular disease and conditions or diseases associated with cardiovascular disease such as hyperlipidemia, atherosclerosis, and metabolic syndrome. Treatment of cardiovascular disease and associated conditions or diseases with the novel MTP inhibitors of the invention, such as miR-30c homologs or miR-30c agonists, reduces MTP-associated lipid production without side effects that occur with other methods of MTP inhibition.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of co-pending applicationhaving U.S. Ser. No. 14/370,846, filed on Jul. 7, 2014, which is a 371of International Application No. PCT/US2013/020718, filed Jan. 9, 2013,and based upon and claiming the benefit of priority from U.S.Provisional Application 61/584,958, filed Jan. 10, 2012, the content ofwhich is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grants DK-46900and HL-95924 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The Sequence Listing in the ASCII text file, named as28080_SequenceListing.txt of 4 KB, created on Dec. 14, 2015, andsubmitted to the United States Patent and Trademark Office via EFS-Web,is incorporated herein by reference.

BACKGROUND OF THE DISCLOSURE

Complications from excess plasma lipid accumulation are one of the mostcommon causes of death in Western societies (1;2) because they enhancerisks for various cardiovascular and metabolic disorders such ashyperlipidemia, atherosclerosis, heart disease, and metabolic syndrome.In plasma, lipids are transported on lipoproteins that provideendogenously produced and dietary lipids to tissues. Plasma lipid levelsare controlled by lipoprotein assembly and their catabolism. Hence,reducing lipoprotein production can be a useful approach to preventand/or treat various cardiovascular and metabolic disorders.

Lipoproteins are synthesized by the intestine and liver using astructural protein, apolipoprotein B (apoB) with the assistance ofmicrosomal triglyceride transfer protein (MTP) (3;4). The assembly ofapoB-containing lipoproteins requires two steps. The first step occurswithin the endoplasmic reticulum that involves the synthesis ofparticles that contain only a small fraction of the lipid core found inthe secreted lipoprotein. A larger core of lipid is added to the nascentparticle in a second step. MTP is considered essential for the transferof various lipids to apoB during the first step of the process.

Pharmacologic inhibition of MTP with Bristol-Myers Squibb's BMS-201038,a potent chemical inhibitor of MTP, reduced low density lipoproteincholesterol (LDL-C) in volunteers with hypercholesterolemia. However,steatorrhea, elevation of serum transaminases and hepatic fataccumulation were observed. Thus, Bristol-Myers Squibb decided thatthese side effects made it unlikely that BMS-201038 could be developedas a drug for large scale use in the treatment of hypercholesterolemia.Combinations using MTP inhibitors and other cholesterol or triglyceridedrugs have been previously disclosed (U.S. Pat. Nos. 6,066,653 and5,883,109) but suffer the same drawbacks as described above for MTPinhibitors used alone. Thus, novel approaches are needed to harnessbeneficial effects of reduced MIT activity.

Hypercholesterolemia is a well-known risk factor for atheroscleroticcardiovascular disease (ASCVD), the major cause of mortality in theWestern world. Numerous epidemiological studies have clearlydemonstrated that pharmacological lowering of total cholesterol (TC) andLow-density Lipoprotein (LDL) Cholesterol (LDL-C) is associated with asignificant reduction in clinical cardiovascular events.Hypercholesterolemia is often caused by a polygenic disorder in themajority of cases and modifications in lifestyle and conventional drugtreatment are usually successful in reducing cholesterol levels.However, in few cases, as in familial hypercholesterolemia, the cause isa monogenic defect and the available treatment in homozygous patientscan be much more challenging and far from optimal because LDL-C levelsremain extremely elevated despite aggressive use of combination therapy.Therefore, for this group of high-risk patients, effective medicaltherapy is urgently needed.

Triglycerides are common types of fats (lipids) that are essential forgood health when present in normal amounts. They account for about 95percent of the body's fatty tissue. Abnormally high triglyceride levelscan result from such causes as cirrhosis of the liver, underactivethyroid (hypothyroidism), poorly controlled diabetes, or pancreatitis(inflammation of the pancreas). Researchers have also identifiedelevated triglycerides as a risk factor for heart disease.

Higher-than-normal triglyceride levels are often associated with knownrisk factors for heart disease, such as low levels of HDL (“good”)cholesterol, high levels of LDL (“bad”) cholesterol and obesity.Triglycerides may also contribute to thickening of artery walls, whichis linked to the development of atherosclerosis.

MicroRNAs (miRs) are small noncoding RNA molecules that can causepost-transcriptional silencing of specific genes, either by theinhibition of translation or through degradation of the targeted mRNA.Since the initial discovery of miRs as regulators of gene expression(11), a role of miRs in development of various diseases such as cancer(12) has been identified. MiRs interact with the 3′-untranslated region(3′-UTR) of target mRNAs and reduce protein synthesis by enhancing mRNAdegradation and/or by interfering with its translation (13). A microRNAcan be completely complementary or can have a region ofnoncomplementarity with a target nucleic acid, consequently resulting ina “bulge” at the region of non-complementarity.

Two miRs have been shown to be involved in lipid metabolism. miR-122 islinked to fatty acid synthesis and oxidation and is currently beingtested as a therapeutic target against hepatitis C infection (1). miR-33regulates expression of ABCA1 and ABCG1, two proteins involved inreverse cholesterol transport (14-18).

A further understanding of the regulation of lipid metabolism by miRscan reveal new physiological mechanisms to reduce lipoproteinproduction, hyperlipidemia and atherosclerosis.

BRIEF SUMMARY OF THE DISCLOSURE

This disclosure provides a novel role for microRNA (miR) regulation oflipid metabolism via the MTP pathway, leading to reductions in apoBsecretion and blood lipid levels. MiR regulation of the MTP pathway isshown herein to reduce hyperlipidemia and atherosclerosis in vivo.Therefore, inhibition of MTP expression and activity by miR regulationis identified as a new therapeutic target for treatment ofcardiovascular disease and conditions or diseases associated withcardiovascular disease such as hyperlipidemia, atherosclerosis, andmetabolic syndrome.

In one embodiment, this disclosure provides a method for treatingcardiovascular disease, hyperlipidemia, atherosclerosis, or metabolicsyndrome in a subject in need thereof, comprising administration of amiR-30c homolog or agonist in an amount effective to treat the diseasein said subject. A particular embodiment further provides administrationof the miR-30c homolog or agonist in combination with at least oneadditional cholesterol reducing agent, such as a statin.

In another embodiment, this disclosure provides a method for reducingserum lipids in a subject in need thereof, comprising administration ofa miR-30c homolog or agonist in an amount effective to reduce serumlipids in said subject. A particular embodiment further providesadministration of the miR-30c homolog or agonist in combination with atleast one additional cholesterol reducing agent, such as a statin.

In an additional embodiment, this disclosure provides a method forreducing MTP activity in a subject in need thereof, comprisingadministration of a miR-30c homolog or agonist in an amount effective toreduce MTP activity in said subject.

In another embodiment, this disclosure provides a pharmaceuticalcomposition for the treatment of cardiovascular disease, hyperlipidemia,atherosclerosis, or metabolic syndrome, comprising an effective amountof a miR-30c homolog or agonist.

In a further embodiment, this disclosure provides a method ofidentifying an agent for the treatment of cardiovascular disease,hyperlipidemia, atherosclerosis, obesity, diabetes, Hepatitis Cinfection, or metabolic syndrome, comprising contacting a cellexpressing a miR-30c homolog with a candidate agent, and assaying theexpression of the miR-30c homolog; wherein an increase in expression ofthe miR-30c homolog identifies the agent as a miR-30c homolog agonistuseful for the treatment of cardiovascular disease, hyperlipidemia,atherosclerosis, or metabolic syndrome.

In another embodiment, this disclosure provides a method of reducinglipid synthesis in a subject in need thereof, comprising administrationof a miR-30c homolog or agonist in an amount effective to reduce lipidsynthesis in said subject.

Another embodiment provides a method of coordinately suppressing lipidsynthesis and lipid secretion by the liver to lower plasma lipids whileavoiding build up of lipids in the liver by administering an effectivedose of miR-30c homolog or agonist in said subject.

BRIEF DESCRIPTION OF THE FIGS.

FIG. 1: Effect of different miR-30s and anti-miR-30s MTP activity andprotein. Huh-7 cells were transfected in triplicate with differentamounts of miR-30b (A), miR-30e (B), anti-miR-30b (C), antagomiR-30e(D), miR-30c (E), anti-miR-30c (F) or a non-specific Scramble miR (Scr)at 100 nM. After 48 hr, cells were used for MTP activity (Bars) andprotein (insets) measurements. Media were used to quantify apoB (G, I)and apoAI (H, J). Data are representative of multiple experiments.

FIG. 2: Regulation of MTP mRNA by miR-30c. (A-C) Huh-7 cells weretransfected with different amounts of miR-30c (A), anti-miR-30c (B), orScr (C). MTP mRNA levels were quantified by qRT-PCR and normalized toARPp0. Values in cells exposed to no miR were normalized to 1 and valuesin other cells are reported relative to this value. (D) Huh-7 cells weretransfected with indicated miRs. After 16 hr, cells were incubated withactinomycin D (10 μg/ml) for different times. MTP/ARPp0 mRNA ratio at 0hr was normalized to 100%. (E-G) COS-7 cells were transfected with 1.5μg of pRc-hMTP in 100 mm tissue culture dishes, a plasmid that expresseshuman MTP using CMV promoter. After overnight incubation, cells weredistributed into different wells of 6-well plates and transfected withScr, miR-30c [30 nM], or anti-miR-30c [30 nM]. After 17 hr, cells wereharvested to measure MTP specific activity (E), protein (F), and mRNA(G). Binding of miR-30c to 3′ -UTR is necessary for MTP mRNAdegradation. MTP sequence in the 3′-UTR was mutated (from GTTTACA inwild type to GAAAACA) pRc-hMTP and transfected in COS-7 cells. After 17h, cells were split into different wells and transfected with Scr miR[50 nM], miR-30c [20 nM], or anti-miR-30c [50 nM] and cells were assayedfor (H) MTP activity after 17 hr. (I-J) Normal (I) or mutated (J) 3′-UTRsequences of MTP were cloned after the stop codon of Renilla luciferasein psiCHECK2 plasmid that also expresses firefly luciferase and thisplasmid was introduced into COS-7 cells. After overnight incubations,cells were transferred to different wells and transfected with Scr [50nM], miR-30c [20 nM], or anti-miR-30c [50 nM]. Luciferase activitieswere assayed after 16 h. Ratios of firefly and Renilla luciferaseactivities were normalized to 100% in cells transfected with Scr.

FIG. 3: Effect of miR-30c on plasma lipids in mice: Male CB57/B16 mice(5/group) were injected (10⁸ infectious units/mouse) retro-orbitallywith lentiviruses that express miR-30c, anti-miR-30c or Scr and startedon a Western diet. Every week mice were fasted for 4 h and blood wascollected to measure total cholesterol (A) and triglyceride (B) for 3weeks. In a separate experiment, at week 5, after blood collection forplasma lipids, mice were injected with P407 and small blood samples werecollected at indicated times to measure cholesterol (C) and triglyceride(D) secretion rates. Slopes between one and 2 h were used to calculateproduction rates. Liver specific MTP knockout mice (L-MTP^(−/−)) wereinjected with miR-30c, anti-miR-30c or Scr expressing lentiviruses andstarted on a Western diet. Plasma was collected at weekly intervals tomeasure changes in triglyceride (E) and cholesterol (F). Plasmatriglyceride (G) and cholesterol (H) were measured on HDL and non-HDLfractions of wild-type C57/B16 mice after 3 weeks. Plasma transaminases,AST (I) and ALT (J) were measured every week for 3 weeks. Hepatic levelsof (K) triglyceride and (L) cholesterol were measured in these livers.

FIG. 4: Effect of miR-30c on atherosclerosis: Female Apoe^(−/−) micewere injected with lentiviruses expressing miR-30c, anti-miR-30c or Scrand started on a Western diet. On week 5, aortic arches were exposed andphotographed (A). Whole aortas (B) and aortic root sections (C) weredissected and stained with Oil red O or Hematoxylin and cosin stain,respectively.

FIG. 5: Expression of miR-30s, MTP and NFY-C in human tissues: (A-C)Abundance of different miRs (A) was measured in indicated human tissuesfrom the First Choice Human Total RNA Survey Panel (Applied Biosystems)by qRT-PCR and normalized to SNORD44. Values in liver were normalized toI. MTP (B) and NFY-C (C) mRNA, and pri-miR-30c (C) levels were assessedby qRT-PCR and normalized to ARPp0. For MTP (B) and pri-miR-30c/NFY-Cvalues in heart and liver, respectively, were adjusted to 1. (D-F) Huh-7cells were transfected with indicated amounts of siDICER duplex andlevels of pri-miR-30c (D), miR-30c (E), and MTP mRNA (F) were measuredafter 18 hr. For pri-miR-30c (D) and MTP (F), ARPp0 served as control.For miR-30c, U6 was used as an endogenous control.*p<0.05;**p<0.01,***p<.001,****p<0.0001; significance calculated byone-way ANOVA.

FIG. S1: miR-30 binding to MTP: (A) TargetScan was used to search formiRs that could interact with the 3′-untranslated region (UTR) of humanMTP mRNA. This search reveals possible binding of several miRs to theMTP transcripts. Next, conservation of the binding sites in variousmammalian MTP 3′-UTR was determined. These studies revealed thatvertebrate MTP mRNAs contain conserved binding sites for the severalmembers of the miR-30 family (a, a-5p, b, b-5p, c, d, and e) andmiR-384-5p. (B) This figure shows putative binding sites for miR-30c indifferent vertebrate MTP mRNA. Pairing site for miR-30c in the 3′-UTR ofvarious mammalian MTP sequences are highlighted in white.

FIG. S2. Conservation of miR-30c in vertebrates: Top line showsschematic representation of different introns and exons in the humanNFY-C gene. miR-30c and miR-30e reside in intron 5 of the gene. Theirlocation is highly conserved in vertebrates.

FIG. S3. Effect of miR-30c and antimiR-30c on plasma lipids andatherosclerosis: Female Apoe^(−/−) mice (n=7/group) were injected withlentiviruses expressing miR-30c or Scr miR and started on a Westerndiet. Plasma cholesterol (A), triglyceride (B), AST (C) and ALT (D) weremeasured weekly. # p<0.05; ## p<0.01, ### p<.001, #### p<0.0001;significance calculated by two-way ANOVA.

FIG. S4. miR-30c is not regulated by common MTP regulators: (A) miR-30cexpression was assessed by qRT-PCR in the livers of MTP^(flox/flox) andMTP liver-specific knockout mice. (B) C57/B16 wild type mice weretreated with WY-14643 compound, a known PPAR-alpha agonist by oralgavage. DMSO was used as a control. (n=4/group) (C) C57/B16 wild-typemice were fed a high fat diet and simultaneously treated withpioglitazone, a known agonist for PPAR-gamma. DMSO was used as acontrol. (n=5/group)

FIG. S5: Lipid biosynthesis processes are targeted by miR-30c: (A)Predicted target genes of miR-30c from TargetScan were used to identifypathways affected using Gene Ontology. This program identified severallipid biosynthetic processes to be targeted by miR-30c. (B) Differentgenes targeted by miR-30c in various processes are listed. (C) Huh-7cells were transfected in triplicate with Scr or miR-30c. After 48 h,mRNAs were isolated to quantify candidate genes and ARPp0 (endogenouscontrol). Ratios in Scr cells were normalized to 1. @ p<0.05; @@ p<0.01,@@@ p<001, @@@@ p<0.0001; significance calculated by student t-test.

DETAILED DESCRIPTION OF THE DISCLOSURE

In the following description, reference is made to the accompanyingdrawings that form a part hereof, and in which is shown by way ofillustration specific embodiments that can be practiced. Theseembodiments are described in detail to enable those skilled in the artto practice the invention, and it is to be understood that otherembodiments can be utilized and that logical changes can be made withoutdeparting from the scope of the present invention. The followingdescription of example embodiments is, therefore, not to be taken in alimited sense, and the scope of the present invention is defined by theappended claims.

The Abstract is provided to comply with 37 C.F.R. §1.72(b) to allow thereader to quickly ascertain the nature and gist of the technicaldisclosure. The Abstract is submitted with the understanding that itwill not be used to interpret or limit the scope or meaning of theclaims.

This disclosure provides a novel role for microRNA (miR) regulation oflipid metabolism via the regulation of lipid synthesis and lipoproteinsecretion pathways, leading to reductions in apoB secretion and bloodlipid levels while avoiding accumulation of lipids in the liver. MiRregulation of the lipid synthesis and lipoprotein secretion pathway isshown herein to reduce hyperlipidemia and atherosclerosis in vivo. Thelipid secretion pathway is affected by reductions in MTP expressionwhereas reductions in lipid synthesis involve several genes such asLPGAT1, ELOVL5, MBOATI. Therefore, inhibition of MTP expression andactivity by miR as well as coordinate down regulation of lipid synthesisis identified as a new therapeutic target for treatment ofcardiovascular disease and conditions or diseases associated withcardiovascular disease such as hyperlipidemia, atherosclerosis, andmetabolic syndrome. Treatment of cardiovascular disease and associatedconditions or diseases with the novel inhibitors of the invention, suchas miR-30c homologs or miR-30c agonists, reduces MTP-associated lipidproduction as well as lipid synthesis via other pathways without sideeffects that occur with other methods of treatment and MTP inhibition.

MTP and lipid metabolism. Microsomal triglyceride transfer protein (MTP)catalyzes the transport of triglyceride, cholesteryl ester, andphosphatidylcholine onto lipoprotein carriers. MTP interacts with lipidsand apoB to transfer lipids to apoB during the assembly ofapoB-containing lipoproteins. In the absence of MTP-mediated lipidtransfer, apoB-containing lipoproteins are not produced, and lipidtransport in the blood is reduced. Thus, inhibition of MTP lowers serumlipids.

As used herein, the term “lipid” or “lipids” refers to lipid moleculesof any type, including fatty acids or acyls such as eicosanoids;glycerolipids such as fats/triglycerides; glycerophospholipids such asphosphatidylcholine; sphingolipids such as ceramides; sterol lipids suchas cholesterol; prenol lipids such as quinines; saccarolipids; andpolyketides.

Inhibition of MTP with reduced side effects. Although reduction oflipoprotein production and transport is desirable for treatment ofelevated blood lipid levels, treatment with previously developed MTPinhibitors resulted in undesirable side effects such as elevated serumtransaminases, hepatic fat accumulation, and steatorrhea, the presenceof excess fat in the stools causing chronic diarrhea. These side effectssuggest possible liver damage as well as intestinal malfunction with theuse of other MTP inhibitors. Therefore, an important aspect of theinvention is the inhibition of MTP and reduction of serum lipid levelswithout these side effects, or where these side effects are reducedrelative to previously reported levels of such side effects using otherMTP inhibitors. This is possible because the MTP inhibitors of theinvention are herein shown to be efficacious in vivo in reducing MTPexpression and serum lipid levels without causing liver damage.

“Inhibition” or “reduction” as described herein includes, for example, a5%, 10%, 25%, 50%, 75%, 80%, 90%, 95%, or even 100% reduction. In someembodiments, MTP activity or expression is reduced or inhibited by 10%,20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%compared to MTP activity or expression in an untreated or controlsubject. Methods thr testing for reduction/inhibition of MTP activity orexpression are known to those of skill in the art and are set forth, forexample, in U.S. Pat. No. 5,789,197. In some embodiments, MTP expressionis reduced or inhibited by miR binding to the MTP mRNA. In otherembodiments, serum or plasma lipid levels in a subject are reduced 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% or morefollowing treatment with the methods and compositions of the invention,compared to lipid levels in the same subject prior to treatment with themethods and compositions of the invention.

The MTP inhibitors of the invention reduce MTP expression and activitywithout negatively affecting liver health or elevating serumtransaminases. Liver health and serum transaminase levels can bedetermined, for example, by measuring levels of aspartate transaminase(AST or SGOT) and/or alanine transaminase (ALT or SGPT). AST and ALT arecommonly measured clinically as a part of diagnostic liver functiontests, to determine liver health. Normal AST ranges=8-40 IU/L (men),6-34 IU/L (women). 5-60 U/L is considered a normal range for ALT,although levels fluctuate over the day for any given individual. The MTPinhibitors of the invention are shown in the Examples below to reduceMTP activity and expression without negatively affecting, for example,AST/ALT levels.

The inventors have discovered that MTP can be inhibited by miRregulation of MTP mRNA. This leads to reduction in lipid transfer ontoapoB, reduced serum cholesterol and lipids, and reduction inatherosclerotic plaque formation, without unwanted side effects of otherinhibitors of MTP.

MTP Inhibitors of the invention. The MTP inhibitors of the inventionencompass microRNA (miR) regulators of MTP expression and activity, andmiR agonists that increase miR expression and activity. As used herein,“inhibiting MTP expression” refers to causing a reduction in MTP proteinlevels. MTP protein levels may be reduced by reducing transcription ofMTP mRNA or pre-mRNA from the MTP gene, such as by preventing binding ofa transcription factor to the MTP genomic locus. MTP protein levels mayalso be reduced by reducing translation of MTP mRNA into MTP protein,such as by preventing binding of a ribozyme to the MTP mRNA, or byincreasing the degradation of MTP mRNA or removal of MTP mRNA from thenucleus. As used herein, “inhibiting MTP activity” refers to causing areduction in at least one MTP activity including, but not limited to,binding lipids, binding apoB, catalyzing transfer of lipids to apoB,facilitating lipoprotein production, increasing apoB lipoproteinsecretion, increasing serum lipid levels, and increasing serumcholesterol and triglyceride levels.

The term “microRNA.” or “miR” refers to a microRNA molecule found ineukaryotes that is involved in RNA-based gene regulation. MiRs interactwith the 3′-untranslated region (3′-UTR) of target mRNAs and can reduceprotein synthesis by enhancing target gene mRNA degradation and/or byinterfering with target gene translation (13). A microRNA according tothe invention can be 18-100 nucleotides in length, and more preferablyfrom 18-80 nucleotides in length. Mature or active miRs can have alength of 19-30 nucleotides, preferably 21-25 nucleotides, particularly21, 22, 23, 24, or 25 nucleotides. The invention also can includedouble-stranded precursors of miRs. MiR precursors typically have alength of about 70-100 nucleotides and have a hairpin conformation. MiRsare generated in vivo from miR precursors by the enzymes Dicer andDrosha, which specifically process long pre-miRNA into functional miRNA.A microRNA can be completely complementary to the target sequence of thetarget mRNA or can have a region of noncomplementarity with a targetmRNA, consequently resulting in a “bulge” at the region ofnon-complementarity.

The active nucleotide miR molecule can be obtained from the miRprecursor through natural processing routes (e.g., using intact cells orcell lysates) or by synthetic processing routes (e.g., using isolatedprocessing enzymes, such as isolated Dicer, Argonaut, or RNAse III). Itis understood that active and precursor miR molecules can also beproduced directly by biological or chemical synthesis by methods knownin the art. When a microRNA is referred to herein by name, the namecorresponds to both the precursor and mature forms, unless otherwiseindicated.

This invention provides miRs useful for inhibiting expression oractivity of MTP. In a particular embodiment, the miR is a miR-30chomolog. By “miR-30c homolog” is meant a nucleic acid molecule thatbinds the 3′ UTR of microsomal triglyceride transfer protein (MTP) mRNA,where such nucleic acid molecule has a nucleobase sequence with at least75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identity to the sequence ofmiR-30c, miR-Base Accession #: M10000736, or a fragment or derivativethereof. Exemplary miR-30c sequences follow:

TABLE 1  pre-miR-30c sequence:ACCAUGCUGUAGUGUGUGUAAACAUCCUACACUCUCAGCUGUGAGCUCAAGGUGGCUGGGAGAGGGUUGUUUACUCCUUCUGCCAUGGA (SEQ ID NO: 1) (hairpin) and-mature sequence: UGUAAACAUCCUACACUCUCAGC (SEQ ID NO: 2).MTP binding domain/seed sequence: 5′-UGUAAAC-3′.

By “miR-30c gene” is meant a polynucleotide that encodes a miR-30chomolog, or analog thereof. By “oligonucleotide” is meant any moleculecomprising a nucleobase sequence. An oligonucleotide may, for example,include one or more modified bases, linkages, sugar moieties, or othermodifications. By “fragment” is meant a portion of a miR molecule. Thisportion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90% of the entire length of the miR nucleic acid molecule.A fragment may contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, or 21 or more nucleotides. By “derivative” is meant anucleic acid that comprises sufficient sequence to bind to and inhibitMTP mRNA.

miR-30c activities include binding to MTP mRNA or the MTP mRNA 3′ UTR,increasing MTP mRNA degradation, inhibiting MTP expression and/oractivity, reducing lipoprotein production, reducing plasma cholesterollevels, reducing plasma triglyceride levels, reducing lipids in plasmaVLDL fraction, reducing or preventing hyperlipidemia, reducing orpreventing obesity, reducing or preventing diabetes, reducing orpreventing atherosclerosis, reducing or preventing atheroscleroticplaque formation, reducing or preventing metabolic syndrome, preventingor reducing Hepatitis C virus infection, and other viral infections thatrequire apoB-lipoproteins for their propagation.

If desired, miR-30c homologs may be modified to stabilize the microRNAagainst degradation, to enhance half-life, or to otherwise improveefficacy. Desirable modifications are described, for example, in U.S.Patent Publication Nos. 20070213292, 20060287260, 20060035254,20060008822, and 20050288244, each of which is hereby incorporated byreference in its entirety.

For increased nuclease resistance and/or binding affinity to the target,the miR-30c homolog can include 2′-O-methyl, 2′-fluorine,2′-O-methoxyethyl, 2′-O-aminopropyl, 2′-amino, and/or phosphorothioatelinkages. Inclusion of locked nucleic acids (LNA), ethylene nucleicacids (ENA), e.g., 2′-4′-ethylene-bridged nucleic acids, and certainnucleobase modifications can also increase binding affinity to thetarget. The inclusion of pyranose sugars in the oligonucleotide backbonecan also decrease endonucleolytic cleavage. An agent can be furthermodified by including a 3′ cationic group, or by inverting thenucleoside at the 3′-terminus with a 3′-3′ linkage. In anotheralternative, the 3′-terminus can be blocked with an aminoalkyl group.Other 3′ conjugates can inhibit 3′-5′ exonucleolytic cleavage. While notbeing bound by theory, a 3′ may inhibit exonucleolytic cleavage bysterically blocking the exonuclease from binding to the 3′ end of theoligonucleotide. Even small alkyl chains, aryl groups, or heterocyclicconjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) canblock 3′-5′-exonucleases. It is understood that the methods and reagentsof the present invention may be used in conjunction with anytechnologies that may be developed to enhance the stability or efficacyof an inhibitory nucleic acid molecule.

miR-30c homologs include nucleobase oligomers containing modifiedbackbones or non-natural internucleoside linkages. Oligomers havingmodified backbones include those that retain a phosphorus atom in thebackbone and those that do not have a phosphorus atom in the backbone.United States patents that teach the preparation ofphosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126;5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and5,625,050, each of which is herein incorporated by reference.

miR-30c homolog nucleobase oligomers having modified oligonucleotidebackbones that do not include a phosphorus atom therein have backbonesthat are formed by short chain alkyl or cycloalkyl internucleosidelinkages, mixed heteroatom and alkyl or cycloalkyl internucleosidelinkages, or one or more short chain hetematomic or heterocyclicinternucleoside linkages. United States patents that teach thepreparation of the above oligonucleotides include, but are not limitedto, U.S. Pat. Nos. 5,470,967; 5,489,677; 5,541,307; 5,561,225;5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070;5,633,360; and 5,677,439, each of which is herein incorporated byreference.

miR-30c homolog nucleobase oligomers may also contain one or moresubstituted sugar moieties. United States patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873;5,670,633; and 5,700,920, each of which is herein incorporated byreference in its entirety.

This disclosure further provides miR-30c agonists to inhibit MTPexpression and/or activity. A “miR-30c agonist” is an agent thatincreases miR-30c activity. An agent that increases miR-30c activitymay, for example, increase the expression of miR-30c, augment miR-30cactivity, or prevent miR-30c inhibition or degradation.

This disclosure further provides nucleic acids encoding a miR-30chomolog, such as the miR-30c gene, or miR-30c agonist, foradministration to a subject in need of treatment according to themethods of the invention.

Treatment of diseases. This disclosure provides methods for treatingcardiovascular disease, hyperlipidemia, atherosclerosis, metabolicsyndrome, and related diseases and conditions in a subject in needthereof, comprising administration of a miR-30c homolog or agonist in anamount effective to treat the disease in said subject.

Further, this disclosure provides methods for reducing serum lipids in asubject in need thereof, comprising administration of a miR-30c homologor agonist in an amount effective to reduce serum lipids in saidsubject.

Additionally, this disclosure provides methods for reducing MTP activityin a subject in need thereof, comprising administration of miR-30c or amiR-30c homolog or agonist in an amount effective to reduce MTP activityin the subject.

The terms “treat,” “treating,” or “treatment” as used herein withregards to a condition refers to slowing the onset or rate ofdevelopment of the condition, delaying the development of symptomsassociated with the condition, reducing or ending symptoms associatedwith the condition, generating a complete or partial regression of thecondition, or some combination thereof. For example, with regard toatherosclerosis, “treatment” can refer to a decrease in the rate ofdevelopment of atherosclerotic plaque deposits, a decrease in the numberor size of existing deposits, or improved plaque stability. Likewise,“treatment” with regard to hyperlipidemia can refer to a decrease inlipid levels, cholesterol levels, and/or triglyceride (TG) levels inblood, serum, or plasma in a treated subject relative to levels in saidsubject prior to treatment. Similarly, “treatment” with regard tocardiovascular disease can refer to an increase in HDL/LDL ratio, or adecrease in blood pressure or hypertension.

As used herein, the terms “prevent” “preventing,” “prevention,”“prophylactic treatment” and the like refer to reducing the probabilityof developing a disorder or condition in a subject, who does not have,but is at risk of or susceptible to developing a disorder or condition.

The subject who is in need of treatment with a miR-30c homolog oragonist can be any animal, including a human. The subject is generallydiagnosed with the condition by skilled artisans, such as a medicalpractitioner. The methods of the invention described herein can beemployed for subjects of any species, gender, age, ethnic population, orgenotype.

The therapeutic methods of the invention include administration of aneffective amount of the agents herein, such as a miR-30c homolog or amiR-30c agonist or a nucleic acid encoding such a miR-30c homolog or amiR-30c agonist to a subject in need thereof.

The term “effective amount,” as used herein, refers to the amountnecessary to elicit the desired biological response. In accordance withthe subject invention, the effective amount of a miR-30c homolog oragonist is the amount necessary to provide an observable effect in atleast one biological factor (i.e., observable decrease in MTP/ApoBlevels) for use in treating a biological condition (such as loweringtotal blood cholesterol levels in a patient diagnosed withhypercholesterolemia). The effective amount can include the amountnecessary to enable a 1% -85% decrease in total serum cholesterol orlipid levels. In certain embodiments, the effective amount enables a 5%,10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, 99% and 100% decrease in severity of complicationsassociated with the biological condition (i.e., complications related tocardiovascular disease or hypercholesterolemia such as obesity, heartdisease, stroke, hypertension, etc.).

Accordingly, the present invention provides methods of treating diseaseand/or disorders or symptoms thereof which comprise administering aneffective amount of a miR-30c homolog or a miR-30c agonist as describedherein to a subject. Thus, one embodiment is a method of treating asubject suffering from or susceptible to a lipid-related disorder, orsymptom thereof. The method includes the step of administering to thesubject an effective amount of a miR-30c homolog or a miR-30c agonist ornucleic acid encoding such a miR-30c homolog or a miR-30c agonist hereinsufficient to treat the lipid-related disease or disorder or symptomthereof, under conditions such that the disease or disorder is treated.

The methods herein include administering to the subject an effectiveamount of a compound described herein, or a composition described hereinto prevent, treat, stabilize, or reduce plasma lipid levels in a subjectin need thereof. Identifying a subject in need of such treatment can bein the judgment of a subject or a health care prossional and can besubjective (e.g. opinion) or objective (e.g. measurable by a test ordiagnostic method).

Conditions treatable. Biological conditions that can be treated via theadministration of a miR-30c homolog or a miR-30c agonist as disclosedherein include, but are not limited to, hypercholesterolemia; hightriglyceride levels (including high LDL levels); obesity, cardiovasculardisease; metabolic syndrome; hypertension; diabetes; as well as anyother symptoms, complications, conditions, or diseases associated withthese diseases. In accordance with the subject invention, theadministration of miR-30c homolog or a miR-30c agonist to a patient candelay or prevent the development of such biological conditions and anyassociated symptoms, complications, conditions, or diseases associatedwith said biological condition.

As used herein, the phrase “disorders associated with hyperlipidemiaand/or hypercholesterolemia” refers to diseases and disorders related toor caused by elevated lipid or cholesterol levels. Such diseases anddisorders include, without limitation, hypercholesterolemia, severehypercholesterolemia, familial combined hyperlipidemia, familialhypercholesterolemia., remnant hyperlipidemia chylomicronemia syndromeand familial hypertriglyceridemia. In some embodiments, the disease issevere hypercholestemlemia. In some embodiments, the disease ishomozygous/heterozygous familial hypercholesterolemia. In someembodiments the disease is hypertriglyceridemia or hyperlipidemia. Insome embodiments the disease is familial combined hyperlipidemia. Insome embodiments disease is viral infections.

As used herein, the term “hyperlipidemia” refers to a pathognomiccondition manifest by elevated serum concentrations of total cholesterol(>200 mg/dL), LDL cholesterol (>130 mg/dL), or triglycerides (>150mb/dL) or decreased HDL cholesterol (<40 mg/dL). Further, as usedherein, “triglycerides” refers to triacylglycerol esters of fatty acids.

The term “cholesterol level” as used herein refers to blood cholesterollevel, serum cholesterol level, plasma cholesterol level, or cholesterollevel from another biological fluid. A decrease in cholesterol levels asused herein may refer to a decrease in total cholesterol levels or adecrease in one or more of total cholesterol, non-HDL cholesterol, LDL,VLDL, and/or IDL levels. A decrease in LDL as used herein may refer to adecrease in total LDL, a decrease in LDL particles, a decrease in smallLDL particles, a decrease in oxidized LDL levels, and/or a decrease inApoB levels. A decrease in VLDL as used herein may refer to a decreasein total VLDL or to a decrease in the level of one or more of VLDLsubparticles V1 to V6. An improvement in HDL/LDL ratio as used hereinrefers to any increase in the ratio of HDL to LDL, and may beaccomplished by decreasing LDL levels, increasing HDL levels, or somecombination thereof. An increase in LDL particle size as used hereinrefers to an increase in mean particle size.

The term “elevated cholesterol level” as used herein refers to acholesterol level that is above an accepted normal threshold level, suchas those promulgated by the National Heart Lung and Blood Institute(NHLBI). The accepted normal threshold cholesterol level may vary fromsubject to subject based on various risk factors, such as for example aprior history of cardiovascular disease (CVD). In certain embodiments, asubject exhibiting elevated cholesterol levels may have a blood LDLlevel greater than or equal to 70 mg/dl. In certain of theseembodiments, a subject exhibiting elevated cholesterol levels may have ablood LDL geater than or equal to 100 mg/dl, in other embodimentsgreater than or equal to 130 mg/dl, in other embodiments greater than orequal to 160 mg/dl, and in still other embodiments greater than or equalto 190 mg/dl. In certain embodiments, a subject exhibiting elevatedcholesterol levels may have a blood total cholesterol level greater thanor equal to 200 mg/dl. In certain of these embodiments, a subjectexhibiting elevated cholesterol levels may have blood total cholesterolgreater than or equal to 240 mg/dl.

The term “triglyceride level” as used herein refers to bloodtriglyceride level, serum triglyceride level, plasma triglyceride level,or triglyceride level from another biological fluid. The term “elevatedtriglyceride level” as used herein refers to a triglyceride level thatis above an accepted normal threshold level. The accepted normalthreshold triglyceride level may vary from subject to subject based onvarious risk factors, such as for example a prior history of CVD. Incertain embodiments, a subject exhibiting elevated triglyceride levelsmay have a blood trialyceride level greater than or equal to 150 mg/dl.In certain of these embodiments, a subject exhibiting elevatedtriglyceride levels may have a blood triglyceride level geater than orequal to 200 mg/dl, in other embodiments greater than or equal to 300mg/dl, in other embodiments greater than or equal to 400 mg/dl, and instill other embodiments greater than or equal to 500 mg/dl.

The present invention provides methods for the treatment and/orprevention of elevated lipids, or for preventing, delaying, and/ortreating the development of elevated lipid-related complications.

In one embodiment, the subject invention provides materials and methodsfor treating and/or preventing high cholesterol or hypercholesterolemia,or for preventing, delaying, and/or treating the development ofhypercholesterolemia (or high cholesterol)-related complications,through the administration of a miR-30c homolog or agonist to a subjectin an amount effective to reduce MTP activity. In another embodiment isprovided materials and methods for treating and/or preventing highplasma lipids or hyperlipidemia, or for preventing, delaying, and/ortreating the development of hyperlipidemia (or high plasmalipid)-related complications, through the administration of a miR-30chomolog or agonist to a subject in an amount effective to reduce MTPactivity.

In some embodiments, one or more of total cholesterol levels, plasmaLDL-cholesterol levels, triglyceride levels, fasting triglycerides (TG)levels, VLDL levels, lipoprotein (a) (Lp(a)) levels, or ApolipoproteinsA-I, A-II, B, and E levels in the subject are reduced by at least 15%,by at least 25%, at least 30%, at least 40%, at least 50%, at least 60%,at least 70%, at least 75%, or at least 80% compared to control bloodlevels.

In some embodiments, triglyceride levels achieved are less than 500mg/dl. In some embodiments, triglyceride levels achieved are less than300 mg/dl. In some enthodiments, triglyceride levels achieved are lessthan 200 mg/dl. In some embodiments, triglyceride levels achieved areless than 150 mg/dl.

In some embodiments, the A,poB/ApoA1 ratio achieved by treatmentaccording to the present invention is from 0.25 to 125. In someembodiments the ApoB/ApoA1 ratio achieved is from 0.1 to 2.0. In someembodiments the apoB level achieved is from 48-130. In some embodimentsthe apoB level achieved is from 20-180.

As used herein, a “control blood level” refers to a level of aparticular blood component in the absence of treatment according to thepresent invention. In some embodiments, the “control blood level” is thelevel of a particular blood component in the subject prior to treatmentof the subject according to the present invention. In some embodiments,the “control blood level” is the level of a particular blood componentif a subject either receiving a placebo or receiving a differenttreatment; e.g. a treatment not including at least three step-wise,increasing dosages of an MTP inhibitor. Reduction of levels of bloodcomponents, including, for example, cholesterol, triglycerides, andapolipoprotein B, can be determined by comparing pre-treatment levels tolevels during or after treatment according to the present invention.

Methods of measuring levels of particular components of blood arewell-known to those of skill in the art. For example, total plasmacholesterol and triglyceride concentrations may be determined by amodification of the Liebermann-Burchard reaction (Abell L L, Levy B B,Brodie B B, Kendall F E. A simplified method for the estimation of totalcholesterol in serum and demonstration of its specificity. J Biol Chem.1952; 195:357-362) and by the method of Kessler and Lederer afterzeolite extraction, (Kessler G, Lederer H. Fluorometric measurement oftriglycerides. In: Skeggs L T, Jr, eds. Automation in AnalyticalChemistry: Technicom Symposia. New York, N.Y.: Madiad Inc;1965:341-344), respectively. Plasma HDL cholesterol may be estimated bythe method of Allain et al (Allain C C, Poon L S, Chan G S G, RichmondW, Fu P C. Enzymatic determination of total serum cholesterol. ClinChem. 1974; 20:470-475) using an enzymatic kit (Biotrol). LDLcholesterol may be calculated using the Freidewald formula. (FreidewaldW T, Levy R I, Fredrickson D S. Estimation of the concentration of lowdensity lipoprotein-cholesterol in plasma without the use of thepreparative ultracentrifuge. Clin Chem. 1972; 18:499-502). Plasma apoB,apoA1, and lipoprotein(a) levels may be measured by immunological assaysas described earlier (Guo H, Chapman M J, Bruckert E, Farriaux J P, DeGennes J L. Lipoprotein Lp(a) in homozygous familialhypercholestemlemia: density profile, particle heterogeneity andapolipoprotein(a) phenotype. Atherosclerosis. 1991; 31:69-83) and basedon laser immunonephelometry (Immuno AG).

“Cardiovascular disease” (CVD) as used herein includes, for example,atherosclerosis, coronary artery disease (CAD), coronary heart disease(CHD), conditions associated with CAD and CHD, cerebrovascular diseaseand conditions associated with cerebrovascular disease, peripheralvascular disease and conditions associated with peripheral vasculardisease, aneurysm, vasculitis, venous thrombosis, and metabolicsyndrome. “Conditions associated with CAD and CHD” as used hereininclude, for example, angina and myocardial infarction (heart attack).“Conditions associated with cerebrovascular disease” as used hereininclude, for example, transient ischemic attack and stroke. “Conditionsassociated with peripheral vascular disease” as used herein include, forexample, claudication. “Conditions associated with CVD” as used hereininclude, for example, low HDL/LDL ratio, and hypertension. Treatment ofcardiovascular disease includes treatment of any one or more of thediseases and conditions as described in this paragraph.

“Arteriosclerosis” or “atherosclerosis” as used herein refers to adisease state characterized by the development and growth ofatherosclerotic lesions or plaque. The identification of those patientswho are in need of treatment for atherosclerosis is well within theknowledge and ability of one skilled in the art. For example, patientswho are either suffering from clinically significant atherosclerosis orwho are at risk of developing atherosclerosis as a result ofhypercholesterolemia are considered patients in need of treatment for acomplication associated with hypercholesterolemina.

Over time, hypercholesterolemia and hypertriglyceridemia can lead to thedevelopment of atheromatous plaques on the inner arterial linings viathe process of atherogenesis, which in turn results in atherosclerosis.Atherosclerosis leads to significantly reduced blood flow through thearteries, which in turn leads to the development of CAD, CHD, andconditions associated with CAD and CHD.

Thus, in a further embodiment is provided materials and methods fortreating and/or preventing atherosclerosis, or for preventing, delaying,and/or treating the development of atherosclerosis or atherosclerosis-related complications, through the administration of an effectiveamount of a miR-30c homolog or agonist to a subject in need thereof. Ina further embodiment is provided materials and methods for treatingand/or preventing cardiovascular disease, or for preventing, delaying,and/or treating the development of cardiovascular disease orcardiovascular disease-related complications, through the administrationof an effective amount of a miR-30c homolog or agonist to a subject inneed thereof.

People with metabolic syndrome are at increased risk of coronary heartdisease and other diseases related to plaque buildup in artery walls(e.g., stroke and peripheral vascular disease) and type 2 diabetes.Metabolic syndrome has become increasingly common in the United Statesand worldwide. It is estimated that over 50 million Americans have riskfactors indicating metabolic syndrome. The dominant underlying riskfactors for this syndrome appear to be abdominal obesity and insulinresistance. Other conditions associated with the syndrome includephysical inactivity, aging, hormonal imbalance and geneticpredisposition.

Metabolic syndrome is a disorder characterized by a group of metabolicrisk factors. These factors include, for example, dyslipidemia,abdominal obesity, elevated blood pressure (hypertension), insulinresistance or glucose intolerance, prothrombotic state, andproinflamtnatory state. Subjects are generally classified as havingmetabolic syndrome if they meet three of the five following criteria: 1)abdominal obesity (waist circumference >35 inches in women, >40 inchesin men); 2) low HDL levels (<50 mg/dL in women, <40 mg/dL in men); 3)high blood pressure (equal to or greater than 130/85 mm Hg) or currenttreatment with antihypertensive medication; 4) hypertriglyceridemia (TGlevels equal to or greater than 150 mg/dL); and 5) impaired fastingglucose (blood glucose levels equal to or greater than 100 mg/dL).Treatment of metabolic disease includes a reduction in at least one ofthese criteria as defined herein.

Administration. One exemplary approach provided by the inventioninvolves administration of a therapeutic miR-30c homolog or agonist,either directly to the site of a potential or actual disease-affectedtissue or systemically (for example, by any conventional administrationtechnique). The dosage of the administered miR-30c homolog or agonistdepends on a number of factors, including the size and health of theindividual patient. For any particular subject, the specific dosageregimes should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the compositions.

A miR-30c homolog of the invention, which may be in the mature orhairpin form, may be provided as a naked oligonucleotide that is capableof entering a cell. In some cases, it may be desirable to utilize aformulation that aids in the delivery of the miR-30c homolog to cells(see, e.g., U.S. Pat. Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798,6,221,959, 6,346,613, and 6,353,055, each of which is herebyincorporated by reference).

In some examples, the miR-30c homolog or agonist composition is at leastpartially crystalline, uniformly crystalline, and/or anhydrous (e.g.,less than 80, 50, 30, 20, or 10% water). In another example, the miR-30chomolog or agonist composition is in an aqueous phase, e.g., in asolution that includes water. The aqueous phase or the crystallinecompositions can be incorporated into a delivery vehicle, e.g., aliposome (particularly for the aqueous phase), or a particle (e.g., amicroparticle as can be appropriate for a crystalline composition).Generally, the miR-30c homolog or agonist composition is formulated in amanner that is compatible with the intended method of administration.

A miR-30c homolog or agonist composition can be formulated incombination with another agent, e.g., another therapeutic agent or anagent that stabilizes an oligonucleotide agent, e.g., a protein thatcomplexes with the oligonucleotide agent. Still other agents includechelators, e.g., EDTA (e.g, to remove divalent cations such asMg.sup.2+), salts, and RNAse inhibitors (e.g., abroad specificity RNAseinhibitor, such as RNAsin).

Polynucleotide therapy featuring a polynucleotide encoding a miR-30chomolog or agonist is another therapeutic approach. Expression vectorsencoding the microRNAs can be delivered to cells of a subject, such ashepatic cells, for inhibition of MTP in the liver. The nucleic acidmolecules must be delivered to the cells of a subject in a form in whichthey can be taken up and are advantageously expressed so thattherapeutically effective levels can be achieved.

Methods for delivery of the polynucleotides to the cell according to theinvention include using a delivery system, such as liposomes, polymers,microspheres, gene therapy vectors, and naked DNA vectors.

Transducing viral (e.g., retroviral, adenoviral, lentiviral andadeno-associated viral) vectors can be used for somatic cell genetherapy, especially because of their high efficiency of infection andstable integration and expression (see, e.g., Cayouette et al., HumanGene Therapy 8:423-430, 1997; Kido et al., Current Eye Research15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649,1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al.,Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). For example, apolynucleotide encoding a miR-30c homolog or agonist can be cloned intoa retroviral vector and expression can be driven from its endogenouspromoter, from the retroviral long terminal repeat, or from a promoterspecific for a target cell type of interest. Other viral vectors thatcan be used include, for example, a vaccinia virus, a bovine papillomavirus, or a herpes virus, such as Epstein-Barr Virus (also see, forexample, the vectors of Miller, Human Gene Therapy 15-14, 1990;Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al.,Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson,Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller etal., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviralvectors are particularly well developed and have been used in clinicalsettings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson etal., U.S. Pat. No.5,399,346).

Non-viral approaches can also be employed for the introduction of amiR-30c homolog to a patient. For example, a miR-30c homolog or agonistcan be introduced into a cell by administering the nucleic acid in thepresence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A.84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham etal., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods inEnzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wuet al., Journal of Biological Chemistry 263:14621, 1988; Wu et al.,Journal of Biological Chemistry 264:16985, 1989), or by micro-injectionunder surgical conditions (Wolff et al., Science 247:1465, 1990).

miR-30c homolog or agonist expression for use in polynucleotide therapymethods can be directed from any suitable promoter (e.g., the humancytomegalovirus (CMV), simian virus 40 (SV40), or metallothioneinpromoters), and regulated by any appropriate mammalian regulatoryelement. For example, if desired, enhancers known to preferentiallydirect gene expression in specific cell types can be used to direct theexpression of a nucleic acid. The enhancers used can include, withoutlimitation, those that are characterized as tissue- or cell-specificenhancers.

In order to realize the therapeutic effect of MTP inhibition, themiR-30c homolog or agonist is administered in an effective amount, alsoreferred to herein as a “therapeutically-effective” amount. As is wellknown in the art, the dosage of the active ingedient(s) significantlydepends on such factors as the method of administration, size of thesubject, and potential side effects. In different embodiments, dependingon these and other factors, a suitable dosage of the active ingredientof the miR-30c homolog or agonist, or miR-30c homolog or agonistcomposition, can be precisely, at least, or no more than, for example, 1mg, 10 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700mg, 800 mg, 900 mg, 1000 mg, 1200 mg, or 1500 mg, or a dosage within arange bounded by any of the foregoing exemplary dosages. Further to theabove embodiments, depending on the same and other factors, thecomposition is administered in the indicated amount by any suitableschedule, e.g., once, twice, or three times a day for a total treatmenttime of one, two, three, four, or five days, and up to, for example,one, two, three, or four weeks. Alternatively, or in addition, thetreatment is administered until a desired amount of MTP inhibition isreached. The desired level of MTP inhibition can be any level deemed bya professional in the medical arts to be appropriate to achieve, and canbe measured directly, by measuring levels of MTP, or indirectly, bymeasuring improvement in one or more symptoms experienced by the subjectin need of treatment.

Generally, doses of active polynucleotide compositions of the presentinvention would be from about 0.01 mg/kg per day to about 1000 mg/kg perday. It is expected that doses ranging from about 50 to about 2000 mg/kgwill be suitable. Lower doses will result from certain forms ofadministration, such as intravenous administration. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day are contemplated to achieve appropriatesystemic levels of the miR-30c homolog or agonist of the invention or ofa polynucleotide encoding such a miR-30c homolog or agonist.

For any particular subject, the specific dosage regimes should beadjusted over time according to the individual need and the professionaljudgment of the person administering or supervising the administrationof the compositions.

Pharmaceutical compositions. This disclosure provides therapeuticcompositions containing a miR-30c homolog or agonist as described hereinfor the treatment or prevention of lipid-associated conditions. In oneembodiment, the present invention provides a pharmaceutical compositionfor the treatment of cardiovascular disease, hyperlipidemia,atherosclerosis, or metabolic syndrome, comprising an effective amountof a miR-30c homolog or agonist. In another embodiment is provided apharmaceutical composition comprising a miR-30c homolog or agonist or anucleic acid molecule encoding a miR-30c homolog or agonist. In anotherembodiment, a miR-30c homolog or agonist or a polynucleotide encodingsuch a miR-30c homolog or agonist, is administered to reduce MTPactivity. Polynucleotides of the invention may be administered as partof a pharmaceutical composition. The compositions should be sterile andcontain a therapeutically effective amount of a miR-30c homolog oragonist or nucleic acid molecule encoding a miR-30c homolog or agonistin a unit of weight or volume suitable for administration to a subject.

A miR-30c homolog or agonist or a nucleic acid molecule encoding amiR-30c homolog or agonist described herein may be administered within apharmaceutically-acceptable diluent, carrier, or excipient, in unitdosage form. Conventional pharmaceutical practice may be employed toprovide suitable formulations or compositions to administer thecompounds to patients suffering from a lipid-associated condition.Administration may begin before the patient is symptomatic. Anyappropriate route of administration may be employed, for example,administration may be parenteral, intravenous, intraarterial,subcutaneous, intratumoral, intramuscular, intracranial, intraorbital,ophthalmic, intraventricular, intrahepatic, intracapsular, intrathecal,intracisternal, intraperitoneal, intranasal, aerosol, suppository, ororal administration. For example, therapeutic formulations may be in theform of liquid solutions or suspensions; for oral administration,formulations may be in the form of tablets or capsules; and forintranasal formulations, in the form of powders, nasal drops, oraerosols.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” Ed. A. R.Gennaro, Lippincourt Williams & Wilkins, Philadelphia, Pa., 2000.Formulations for parenteral administration may, for example, containexcipients, sterile water, or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds. Otherpotentially useful parenteral delivery systems for inhibitory nucleicacid molecules include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Formulationsfor inhalation may contain excipients, for example, lactose, or may beaqueous solutions containing, for example, polyoxyethylene-9-laurylether, glycocholate and deoxycholate, or may be oily solutions foradministration in the form of nasal drops, or as a gel.

The formulations can be administered to human patients intherapeutically effective amounts depending on such variables as thetype and extent of the disorder, the overall health status of theparticular patient, the formulation of the compound excipients, and itsroute of administration.

Combination therapy—The compositions herein are further provided incombination with one or more additional therapeutic means to treatcardiovascular disease, hyperlipidemia, atherosclerosis, or metabolicsyndrome, for example by reducing plasma lipid and cholesterol levels,and/or reducing or reversing atherosclerotic plaque formation.

Reducing fat and cholesterol intake. This disclosure provides foradministration of an effective amount of the compositions of theinvention in combination with a reduced fat, reduced cholesterol diet.The primary means of reducing or eliminating dietary cholesterol andlipid intake occurs through changes in food intake. Total dietarycholesterol is preferably limited to below 200 mg per day, even morepreferably below 100 mg per day. A patient is assigned a diet in whichhigh-fat and high-cholesterol foods (such as egg yolks, high fat dairyproducts such as whole milk, organ meats, and pastries) and saturatedfats are to be significantly reduced, and are preferably eliminatedentirely. Saturated fatty acids are precursors for cholesterol synthesisand therefore, the level of their intake is positively associated withhigher cholesterol level. Major food sources of saturated fat are animalfood-based products (bacon, lard, butter, etc.) and fried foods.

It is recommended that the patient substitute unsaturated fats forsaturated fats. Replacement of foods high in saturated fatty acids withpolyunsaturated or monounsaturated fat rich foods reduces serumcholesterol levels. Unsaturated fat includes polyunsaturated fat andmonounsaturated fat, both of which are predominantly found in plantproducts. Examples of polyunsaturated fat food sources include soybean,sunflower, fish and corn oils. Monounsaturated fat is found in highcontent in olive, peanut, and canola oils.

High fiber foods such as unprocessed whole grains, vegetables, andfruits, are encouraged. Foods rich in fiber, both soluble and insolublefiber, prevent the re-absorption of cholesterol-rich bile acids from thesmall intestines back into circulation, thereby reducing circulatingcholesterol. Thus, high-fiber, unprocessed foods inhibit cholesterolabsorption and lower cholesterol in the body. The patient may alsoconsume fiber supplements, such as Metamucil™ or Benefiber™, to increasedietary fiber intake.

Food containing phytosterols are further encouraged. Phytosterols aresterol compounds produced by plants which, because they are structurallyvery similar to cholesterol, inhibit cholesterol uptake in the digestivesystem. Important sources of phytosterols are rice bran, corn bran, corngerm, wheat germ oil, corn oil, safflower oil, olive oil, cotton seedoil, soybean oil, e.g., soybean oil distillates, peanut oil, black tea,orange juice, green tea, kale, broccoli, sesame seeds, shea oils,grapeseed oil, rapeseed oil, linseed oil, and canola oil.

Prevention of dietary cholesterol absorption in the intestines may beaugmented by treating the patient with a cholesterol absorptioninhibitor (CAI). CAIs include, for example, ezetimibe;1,4-Diphenylazetidin.-2 -ones; 4-biarylyl-1. -phenylazetidin-2-ones;4-(hydroxyphenyl)azetidin-2-ones;1,4-diphenyl-3-hydroxyalkyl-2-azetidinones;4-biphenyl-1-phenylazetidin-2-ones; 4-biarylyl-1-phenylazetidin-2-ones;and 4-biphenylylazetidinones. Another class of CAIs are bile acidsequestrants, such as cholestyramine, colesevelam and colestipol.

Blocking de novo cholesterol synthesis. This disclosure further providesfor administration of an effective amount of the compositions of theinvention in combination with at least one additional cholesterolreducing agent. Such agents reduce serum cholesterol by partially orcompletely blocking de novo cholesterol synthesis. Cholesterol reducingagents encompass several classes of drugs that include HMG CoA reductaseinhibitors (statins), γ-tocotrienol, bisphosphonates,cholesterol-ester-transfer-protein (“CETP”) inhibitors, squalenesynthase inhibitors, soluble guanylate cyclase modulators (“sGCmodulators”), nicotinic acid, and derivatives thereof (e.g. AGI-1067).In a preferred embodiment, the cholesterol reducing agent is a statin.

Statins, γ-tocotrienol, and bisphosphonates inhibit the mevalonate tocholesterol conversion pathway. Statins and γ-tocotrienol inhibitHMG-CoA reductase, a rate-limiting enzyme necessary for cholesterolproduction, and decrease the production of mevalonate and subsequentproducts on the way to construction of the cholesterol molecule. Statintherapy has been demonstrated to provide significant reductions in serumcholesterol levels. For example, administration of atorvastatin 80 mgdaily significantly lowers plasma cholesterol concentrations. Statinsinclude, but are not limited to, atorvastatin (Lipitor®, bervastatin,carvastatin, crilvastatin, dalvastatin, fluvastatin (Lescol®),glenvastatin, fluindostatin, velostatin, lovastatin (mevinolin;Mevacor®), pravastatin (Pravachol®), rosuvastatin (Crestor®), andsimvastatin (Zocor®). Statins identical to lovastatin and itsderivatives can be produced by a variety of filamentous fungi, includingMonascus, Aspergillus, Penicillium, Pleurotus, Pythium, Hypomyces,Paelicilomyces, Eupenicillium, and Doratomyces (Manzoni M, Rollini M.,Appl Microbiol Biatechnol. 58:555-64, 2002).

Bisphosphonates (such as clodronate and etidronate) that closelyresemble pyrophosphate—a normal byproduct of human metabolism—areincorporated into adenosine triphosphate (ATP) analogues. The newestgeneration of bisphosphonates, which contain nitrogen (such aspamidronate, alendronate, risedronate, and ibandronate), are believed toinhibit post-translational modification within the mevalonate pathway.

A cholesterol reducing agent can be orally administered in the form of asublingual tablet, buccal tablet, extended-release (long-acting)capsule, or spray. For a statin, about 2 mg to 80 mg, about 5 mg to 40mg, or about 10 to 80 mg of a statin per day for an adult can be orallyadministered. For a cholesterol absorption inhibitor (e.g. ezetimibe),about 2 mg to 80 mg, about 5 mg to 40 mg, or about 10 to 80 mg of acholesterol absorption inhibitor per day for an adult can be orallyadministered. For a bile acid sequestrant (e.g. cholestyramine,colesevelam or colestipol), about 1 g to 30 g, about 0.2 g to 6 g, about0.1 g to 3 g, about 0.02 g to 0.6 g, about 0.01 g to 0.3 g, about 5 g to150 g, about 2 g to 60 g or about 10 g to 300 g of a bile acidsequestrant per day for an adult can be orally administered.

This disclosure further provides kits for the treatment or prevention ofcardiovascular disease, hyperlipidemia, atherosclerosis, or metabolicsyndrome. In one embodiment, the kit provides a miR-30c homolog oragonist, or a nucleotide encoding a miR-30c homolog or agonist, foradministration to a subject. The kit can also be useful in evaluatingefficacies in cell culture and animal models.

Screening Assays. This disclosure further provides a method ofidentifying an agent for the treatment of cardiovascular disease,hyperlipidemia, atherosclerosis, or metabolic syndrome, the methodinvolving contacting a cell expressing a miR-30c homolog with acandidate agent; and assaying the expression of a miR-30c homolog, wherean increase in the a miR-30c homolog expression identifies the agent asa miR-30c homolog agonist useful for the treatment of cardiovasculardisease, hyperlipidemia, atherosclerosis, or metabolic syndrome.

One embodiment of the invention encompasses a method of identifying anagent that increases the expression or activity of a miR-30c homolog.Accordingly, compounds that increase the expression or activity of amicroRNA of the invention or a variant, or portion thereof are useful inthe methods of the invention for the treatment or prevention ofcardiovascular disease, hyperlipidemia, atherosclerosis, or metabolicsyndrome. Any number of methods are available for carrying out screeningassays to identify such compounds. In one approach, the method comprisescontacting a cell that expresses miR-30c with an agent and comparing thelevel of expression in the cell contacted by the agent with the level ofexpression in a control cell, wherein an agent that increases theexpression of a microRNA of the invention thereby inhibits MTP and isidentified as an agent for the treatment or prevention of cardiovasculardisease, hyperlipidemia, atherosclerosis, or metabolic syndrome.

In other embodiments, the agent acts as a microRNA mimetic, whichsubstantially fulfills the function of an microRNA of the invention.Candidate mimetics include organic molecules, peptides, polypeptides,nucleic acid molecules. Small molecules of the invention preferably havea molecular weight below 2,000 daltons, more preferably between 300 and1,000 daltons, and still more preferably between 400 and 700 daltons. Itis preferred that these small molecules are organic molecules. Compoundsisolated by any approach described herein may be used as therapeutics totreat cardiovascular disease, hyperlipidemia, atherosclerosis, ormetabolic syndrome.

Compounds that increase the expression of a microRNA of the inventionare also useful in the methods of the invention. Any number of methodsare available for carrying out screening assays to identify newcandidate compounds that increase the expression of miR-30c. Theinvention also includes novel compounds identified by theabove-described screening assays. Optionally, such compounds arecharacterized in one or more appropriate animal models to determine theefficacy of the compound for the treatment of cardiovascular disease,hyperlipidemia, atherosclerosis, or metabolic syndrome. Desirably,characterization in an animal model can also be used to determine thetoxicity, side effects, or mechanism of action of treatment with such acompound. Furthermore, novel compounds identified in any of theabove-described screening assays may be used for the treatment ofcardiovascular disease, hyperlipidemia, atherosclerosis, or metabolicsyndrome in a subject. Such compounds are useful alone or in combinationwith other conventional therapies known in the art.

Test Compounds and Extracts. In general, compounds capable of treatingcardiovascular disease, hyperlipidemia, atherosclerosis, or metabolicsyndrome by increasing the expression or biological activity of amiR-30c homolog are identified from large libraries of either naturalproduct or synthetic (or semi-synthetic) extracts or chemical librariesaccording to methods known in the art. Numerous methods are alsoavailable for generating random or directed synthesis (e.g.,semi-synthesis or total synthesis) of any number of chemical compounds,including, but not limited to, saccharide-, lipid-, peptide-, andnucleic acid-based compounds. Synthetic compound libraries arecommercially available from Brandon Associates (Merrimack, N.H.) andAldrich Chemical (Milwaukee, Wis.). Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant, and animal extractsare commercially available from a number of sources, including Biotics(Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute(Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.).

In one embodiment, test compounds of the invention are present in anycombinatorial library known in the art, including: biological libraries;peptide libraries (libraries of molecules having the functionalities ofpeptides, but with a novel, non-peptide backbone which are resistant toenzymatic degradation but which nevertheless remain bioactive; see,e.g., Zuckermann, R. N. et al., J. Med. Chem. 37:2678-85, 1994);spatially addressable parallel solid phase or solution phase libraries;synthetic library methods requiring deconvolution; the ‘one-beadone-compound’ library method; and synthetic library methods usingaffinity chromatography selection. The biological library and peptoidlibrary approaches are limited to peptide libraries, while the otherfour approaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, Anticancer Drug Des. 12:145,1997).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci.U.S.A. 90:6909, 1993; Etb et al., Proc. Natl. Acad. Sci. USA 91:11422,1994; Zuckermarm et al., J. Med. Chem. 37:2678, 1994; Cho et al.,Science 261:1303, 1993; Carrell et al., Angew. Chem. Int. Ed. Engl.33:2059, 1994; Care11 et al., Angew. Chem. Int. Ed. Engl. 33:2061, 1994;and Gallop et al., J. Med. Chem. 37:1233, 1994.

Libraries of compounds may be presented in solution (e.g., Houghten,Biotechniques 13:412-421, 1992), or on beads (Lam, Nature 354:82-84,1991), chips (Fodor, Nature 364:555-556, 1993), bacteria (Ladner, U.S.Pat. No. 5,223,409), spores (Ladner, U.S. Pat. No. 5,223,409), plasmids(Cull et al., Proc Natl Acad Sci USA 89:1865-1869, 1992) or on phage(Scott and Smith, Science 249:386-390, 1990; Devlin, Science249:404-406, 1990; Cwirla et al. Proc. Natl. Acad. Sci. 87:6378-6382,1990; Felici, J. Mol. Biol. 222:301-310, 1991; Ladner supra.). In anembodiment of the invention, a high thoroughput approach can be used toscreen different chemicals for their potency to enhance the activity ofmiR-30c.

Those skilled in the field of drug discovery and development willunderstand that the precise source of a compound or test extract is notcritical to the screening procedure(s) of the invention. Accordingly,virtually any number of chemical extracts or compounds can be screenedusing the methods described herein. Examples of such extracts orcompounds include, but are not limited to, plant-, fungal-, prokaryotic-or animal-based extracts, fermentation broths, and synthetic compounds,as well as modification of existing compounds.

When a crude extract is found to enhance the biological activity ofmiR-30c, variant, or fragnent thereof, further fractionation of thepositive lead extract is necessary to isolate chemical constituentsresponsible for the observed effect. Thus, the goal of the extraction,fractionation, and purification process is the careful characterizationand identification of a chemical entity within the crude extract havingmiR-30c agonistic or MTP-inhibiting activity. Methods of fractionationand purification of such heterogeneous extracts are known in the art. Ifdesired, compounds shown to be useful miR-30c agonists and/or MTPinhibitors are chemically modified according to methods known in theart.

The present description is further illustrated by the followingexamples, which should not be construed as limiting in any way. Thecontents of all cited references (including literature references,issued patents, published patent applications as cited throughout thisapplication) are hereby expressly incorporated by reference.

EXAMPLES

Identification of MTP regulation by miR-30c. TargetScan algorithm basedon seed recognition identified several miRs that interact with the humanMTP transcript. Evolutionary conservation studies among vertebrates,however, narrowed this list to several members of the miR-30 family(FIG. S1A). The miR-30 seed recognition site is conserved in differentmammalian MIP mRNAs (FIG. S1B). To examine effects on MTP expression,these miRs were introduced into human hepatoma Huh-7 cells that expressMTP and secrete apoB. miR-30b (FIG. 1A), miR-30c (FIG. 1B), antagomiR(anti-miR)-30b (FIG. 1C) and anti-miR-30e (FIG. 1D) had no significanteffect on MTP specific activity. Similarly a nonspecific scramble miR(Scr) had no effect compared to no miR (FIG. 1). But miR-30csignificantly decreased (˜50%) MTP activity and protein levels (FIG.1E). In particular, miR-30c decreases MTP activity by approximately 50%.MicroRNA-30c mimic (obtained from Dhatmacon was overexpressed in Huh-7hepatoma cells and incubated for approximately 48 hours. Afterward, MTPactivity was assayed using a kit from Chylos, Inc. Effect of MTPactivity was compared to a scramble miR that was also transfected inHuh-7 cells in parallel. Further, a miR-30c chemical antagonist,anti-miR-30c, modestly increased MTP protein and activity at higherconcentrations compared with Scr (FIG. 2F). This suggested that miR-30creduces MTP expression, although the modest increase seen withanti-miR-30c suggests that this antagonist is not a potent MTP inducer.

Regulation of MTP and apoB synthesis by miR-30c is independent of apoAI.Since MTP is a critical chaperone for the biosynthesis ofapoB-lipoproteins, the inventors studied the effect of miR-30c apoBsecretion in Huh-7 cells. Increasing concentrations of miR-30c decreasedapoB (FIG. 1G), but not apoAI (FIG. 1H), secretion. Anti-miR-30c [100nM] increased apoB secretion by 6- to 10-fold (FIG. 1I) but had noeffect on apoAI secretion (FIG. 1J). Scr miR had no effect on apoB andapoAI secretion. These studies indicate that miR-30c reduces MTPexpression and apoB-lipoprotein secretion I secretion.

miR-30c reduces MTP protein and activity by targeting MTP mRNA. Tounderstand how miR-30c regulates MTP protein and activity, the inventorsmeasured mRNA levels in cells transfected with miR-30c and anti-miR-30c.Increasing concentrations of miR-30c decreased MTP mRNA by 60-70% (FIG.2A); however, different concentrations of anti-miR-30c (FIG. 2B) and ScrmiR (FIG. 2C) had no significant effect. The absence of specific effectwith anti-miR-30c might be related to low expression of miR-30c in thesecells. These studies indicated that miR-30c acts by decreasing MTP mRNA.

miR-30c reduces MTP RNA by increasing the rate of MTP RNA degradation.Attempts were then made to understand how miR-30c reduces MTP mRNA.Cells were treated with actinomycin D to inhibit gene transcription andstudied time dependent changes in MTP mRNA (FIG. 2D). The inventorsobserved that MTP mRNA was removed faster in cells expressing miR-30cthan those expressing either Scr, anti-miR-30c or no miR. Mechanisms ofMTP mRNA regulation by miR-30c were further evaluated in COS-7 cellsthat do not express MTP. MTP activity (FIG. 2E), protein (FIG. 2F) andmRNA (FIG. 2G) were reduced when MTP expression plasmids wereco-transfected with miR-30c compared to Scr. In contrast, anti-miR-30cincreased MTP expression. Thus, miR-30c reduces MTP RNA by increasingthe rate of MTP RNA degradation.

miR-30c binds to the 3′-UTR of MTP mRNA to induce MTP mRNA degradation.Next, the inventors tested the hypothesis that seed sequences in the3′-UTR of MTP are necessary for mRNA degradation by miR-30c. The miR-30ctarget sequence is located between 89-95 bases from the stop codon ofthe human MTP mRNA. The inventors found that miR-30c and its bindingsite in the 3′-UTR. of MTP are conserved in vertebrates (FIG. S1B). Thisrelationship suggests that miR-30c and MTP might have co-evolved andthis conservation serves an important function. Mutagenesis of the seedsequence in the 3′-UTR of human MTP abolished miR-30c dependent decreasein MTP activity (FIG. 2H). The importance of 3′-UTR was furtherevaluated using a psiCHECK2 plasmid encoding a luciferase gene with the3′-UTR of MTP (FIG. 2I-J). In the presence of miR-30c, the luciferaseactivity decreased compared to the Scr miR. By contrast, the luciferaseactivity increased in cells expressing anti-miR-30c (FIG. 2I). When the3′-UTR was mutated in this construct the effects of miR-30c andanti-miR-30c were abolished (FIG. 2J). Therefore, miR-30c binds to the3′-UTR of MTP and induces mRNA degradation leading to reduced MTPactivity and apoB secretion.

miR-30c reduces cholesterol and triglycerides in VLDL/LDL plasmafractions in mice fed standard Western diet. The inventors then exploredin vivo physiologic consequences of miR-30c expression. The inventorsstudied the effect of different miRs on plasma lipoproteins, AST(aspartate aminotransferase) and ALT (alanine aminotransferase) levelsas well as hepatic lipids. C57/B16 mice were transduced withlentiviruses expressing different miRs and provided with ad libitumWestern diet. After 3 weeks, hepatic levels of miR-30c wereapproximately 4-fold higher, compared with Scr mice. miR-30c reducedcholesterol (FIG. 3H) in VLDL/LDL fractions but had no effect ontriglyceride (3G), plasma AST (FIG. 3I) and ALT (FIG. 3.J) levels.Furthermore, there were no significant differences in hepatictriglyceride (FIG. 3K) and cholesterol (FIG. 3L) in these mice.

miR-30c reduces plasma cholesterol and plasma triglycerides leading toreduced hyperlipidemia. Plasma cholesterol were significantly lower inmice injected with miR-30c mimic and higher in mice expressinganti-miR-30c compared with control mice injected with Scr (FIG. 3A). Theamounts of total plasma triglyceride in these animals showed a trendsimilar to the cholesterol (FIG. 3B). ApoB lipoprotein precipitationanalysis of the plasma showed that miR-30c mainly reduced lipids innon-HDL fractions (FIG. 3H). miR-30c had no effect on liver lipids (FIG.3K-L) and plasma AST/ALT (FIG. 3I-J), indicating no negative impact onliver health in treated animals. These studies suggest that miR-30cprevents increases in Western diet induced hyperlipidemia whereasanti-miR-30c augments these changes.

miR-30c reduces hyperlipidemia by reducing hepatic lipoproteinproduction. To understand mechanisms for reduced hyperlipidemia inmiR-30c expressing mice, the inventors studied hepatic lipoproteinproduction in Western diet fed mice after the inhibition of lipases byP407 to prevent lipoprotein catabolism. Triglyceride production rateswere significantly higher in anti-miR-30c (372 mg/dl/h), and lower inmiR-30c (119 mg/dl/h) compared with Set (205 mg/dl/h) expressing mice(FIG. 3D). Cholesterol production rates were not affected in miR-30cexpressing mice and were increased in anti-miR-30c expressing mice (FIG.3C). These studies indicate that reduced hyperlipidemia in miR-30cexpressing mice might b secondary to decreased hepatic lipoproteinproduction.

miR-30c reduction of plasma lipids occurs specifically through MTP. miRsmay regulate expression of several genes and modulate multiple pathways.Therefore, the inventors evaluated the specific need of MTP in thereduction of hyperlipidemia by miR-30c by transducing liver-specific MTPknockout (Alb-Cre-Mttp^(fl/fl), L-MTP^(−/−)) mice with lentivirusesexpressing different miRs. miR-30c and anti-miR-30c did not affectplasma triglyceride (FIG. 3E) and cholesterol (FIG. 3F) in L-MTP^(−/−)mice, indicating that reductions in plasma lipids by miR-30c requiresMTP expression.

miR-30c targets more than one gene involved in lipid metabolism. Tounderstand reasons for decreased lipid synthesis, Gene Ontology analysiswas performed for miR-30c target genes. This analysis revealed thatmiR-30c is predicted to affect several pathways in lipid metabolism,such as pathways for biosynthesis of steroids, glycerolipids,phospholipids, and fatty acids (FIG. S5A), by targeting several genesinvolved in these pathways (FIG. S5B). The inventors found thatexpression of miR-30c in Huh-7 cells reduced mRNA levels of several ofthese target genes, including LPGAT1, ELOVL5, MBOAT1, and StARD3 (FIG.S5C). Thus, miR-30c appears to coordinately decrease lipid biosynthesisand lipoprotein assembly/secretion to avoid steatosis and lower plasmalipids.

miR-30c prevents progression of atherosclerosis. Female Apoe^(−/−) icewere injected with different miRs and fed a Western diet for 5 weeks(FIG. 4). Visualization of atherosclerotic plaques at the aortic arch onweek 7 indicated that miR-30c expressing mice had smaller lesionscompared to control mice injected with Scr (FIG. 4A). In contrast, miceexpressing anti-miR-30c had more lesions. Additionally, whole aortaswere stained with Oil Red O (FIG. 4B). The lipid staining was less inmiR-30c and more in anti-miR-30c expressing mice compared with Scr mice.Similarly, Hetnatoxylin and eosin stain of the aortic sections revealsmall plaque sizes in miR-30c group. (FIG. 4C). Similar results wereobtained in a different experiment where miRs were injected. Plasmacholesterol and triglyceride were measured in Apoe^(−/−) mice for 6weeks (FIG. S3A-B). In addition, plasma transaminases (AST and ALT) werealso measured (FIG. S3C-D). Virus was injected into the retro-orbitalpart of the eye in mice. All animal handling measures and viralinjections were done according to and approved by SUNY Downstate AnimalCare Facility. Immediately afterward, mice were started on a Westerndiet. These studies indicate that miR-30c protects against, andanti-miR-30c augments, atherosclerosis in Apoe^(−/−) mice.

miR-30c is regulated by post-transcriptional processing of miR-30c mRNA.Experiments were then performed to understand the regulation of miR-30csynthesis. Tissue expression studies revealed that miR-30b, miR-30c andmiR-30e (FIG. 5A) are highly expressed in human heart and skeletalmuscle. Notably, their levels were low in the intestine and liver,tissues that highly express MTP (FIG. 5B), indicating a negativerelationship between these miRNAs and MTP. miR-30c resides in the intron5 of the human gene nuclear transcription factor Y subunit C (NFY-C) andis conserved in vertebrates (FIG. S2). INFY-C was ubiquitously expressedwith high levels in the heart and testes (FIG. 5C). This expressionpattern was very different than that of miR-30c (FIG. 5A). Differentsteady state levels of the host gene and intronic miR-30c indicated thattheir expression might be regulated differently involving transcriptionor post-transcriptional mechanisms.

To further identify mechanisms for regulation of miR-30c, the inventorsmeasured expression of pri-miR-30c in human tissues. Pri-miR-30cexpression levels were similar to those of NFY-C indicating that bothare transcribed simultaneously and tissue levels of miR-30c might beregulated at posttranscriptional levels (FIG. 5C). To test thishypothesis, Huh-7 cells were treated with siDICER and the amounts ofpri-miR-30c, miR-30c, and MTP transcripts were measured. siDICER reduceddicer mRNA levels by 90%. siDICER had no significant effect onpri-miR-30c (FIG. 5D) but reduced miR-30c levels (FIG. 5E). Moreover,siDICER increased MTP mRNA (FIG. 5F). The inventors interpret these datato suggest that NFY-C and pri-miR30c are co-transcribed and the steadystate tissue levels of NFY-C and miR-30c differ mainly due topost-transcriptional processing of pri-miR-30c. Reductions in miR-30caccompany increases in MTP mRNA levels. MicroRNAs that potentially bindto MTP mRNA were analyzed using a database called TargetScan. Theresults of that analysis revealed that a family of microRNAs (miR-30)potentially bind to MTP mRNA 3′ UTR. The binding site on MTP mRNA 3′ UTRis also evolutionary conserved in vertebrates (FIG. S1). MicroRNA-30c,in and of itself, is also conserved in vertebrates as well (FIG. S2).

Regulation of miR-30c occurs independently of changes in plasma lipidlevels and other mechanisms that regulate MTP expression. The inventorsthen considered the possibility that MTP might reciprocally regulatemiR-30c expression and measured miR-30c levels in the livers ofMttp^(fl/fl) and L-MTP^(−/−) mice (FIG. S4A). miR-30c levels weresimilar in the liver of these mice suggesting that MTP expression doesnot affect miR-30c expression. Next, the inventors hypothesized thatmiR-30c might be regulated by changes in plasma lipid levels. However,treatment with a PPARα agonist, or treatment with a PPAR-gamma agonistsimultaneously on a Western diet had no effect on miR-30c levels (FIG.S4B-C). Therefore, miR-30c is not regulated by several common mechanismsknown to regulate MTP expression. Identification of additional pathwaysand modifiers that regulate miR-30c can elucidate additional componentsinvolved in lipid metabolism.

miR-30c reduces MTP activity without side effects associated with MTPinhibition. These studies provide evidence that miR30c enhancesdegradation of MTP mRNA and reduces protein and activity resulting inreduced production of apoB-lipoproteins and lowering of plasma lipids.No side effects associated with other forms of MTP inhibition werenoted, possibly because low levels of MTP reduction is effective forbeneficial effect without causing liver damage. Hence, treatment withmiR-30c is found to be a novel approach to lower plasma lipids.

miR-30c antagonist increases ApoB secretion. Anti-miR-30c is expected tobind endogenous miR-30c and reverse its effects. In this study,anti-miR-30c modestly increased MTP mRNA but highly increased apoBsecretion in Huh-7 cells (FIG. 1) and in mice (FIG. 3). However, it hadmodest effect on plasma lipids in L-MTP^(−/−) mice. The modest effectsof anti-miR-30c on MTP expression might be secondary to low levels ofmiR-30c expression. However, stronger effects on apoB secretion suggestthat anti-miR-30c might, besides affecting MTP expression, also affectadditional proteins that are critical for apoB secretion. Blast analysisrevealed that anti-miR30c could potentially bind to miR-30b andtherefore, it might provide additional protection against apoBdegradation and promote its secretion. However, it should be pointed outthat the anti-miR-30c effects do require MTP expression as its effectson apoB levels were not seen in L-MTP^(−/−) mice. Therefore,anti-miR-30c could affect apoB levels by two mechanisms; one involvesits inhibition of miR-30c and the other involves inhibition of anotherprotein/mechanism leading to synergistic increase in apoB secretion.

Specificity of miR-30c for MTP. All the miR-30 family members containthe same seed sequence (FIG. S1). They differ with regards to thecompensatory binding sites. MiR-30c has the maximum compensatory bindingsites in the MTP transcript. The usage of the phrase ‘compensatorybinding sites’ refers to the miR-30c sequence in and of itself. Asdefined earlier in this document, microRNA-30c contains a seed sequencethat is evolutionary conserved. It is thought that the seed sequence hasstrongest affinity for its target mRNA. The compensatory binding sitesonly assist and improve the fidelity of the microRNA to its target. Withthat said, microRNAs do not have 100% fidelity to its target bindingsite. However, increased compensatory binding sites implies increasedfidelity for its target; hence, better binding and targeting.Compensatory binding sites are any number of bases downstream from theseed sequence. The maximum number of compensatory binding sites islimited to the sequence length of the microRNA itself. For example, if amicroRNA has 100% sequence complementarity to its target, one can saythat all compensatory binding sites are used. Although it is possiblethat other miR-30 family members might affect MTP expression underdifferent experimental conditions, the inventors interpret these resultsto suggest that, besides the seed sequence, compensatory binding sitesmight also play a role in mRNA degradation. Possibly, specificity ofmiRs to bind a particular mRNA target can be enhanced by increasing thefidelity of their binding to these compensatory binding sites.

In summary, the inventors have provided evidence that miR-30c reducesMTP mRNA, protein and activity. Further, the inventors have shown thatMTP mRNA is degraded faster in the presence of miR-30c. This accelerateddegradation is due to the binding of miR-30c to the of MTP. Further, theinventors have shown that miR-30c lowers plasma triglyceride andcholesterol levels by reducing triglyceride production rates inhyperlipidemia mice. In another study the inventors observed thatatherosclerotic plaques are smaller in Apoe^(−/−) mice expressingmiR-30c. Taken together, the inventors have provided evidence thatmiR-30c reduces plasma lipids and atherosclerosis by reducing MTPexpression. High fiber foods such as unprocessed whole grains,vegetables, and fruits, are encouraged. Foods rich in fiber, bothsoluble and insoluble fiber, prevent the re-absorption ofcholesterol-rich bile acids from the small intestines back intocirculation, thereby reducing circulating cholesterol. Thus, high-fiber,unprocessed foods inhibit cholesterol absorption and lower cholesterolin the body. The patient may also consume fiber supplements, such asMetamucil™ or Benefiber™, to increase dietary fiber intake.

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1. -8. (canceled)
 9. A pharmaceutical composition for the treatment ofcardiovascular disease, hyperlipidemia, atherosclerosis, obesity,diabetes, or metabolic syndrome, comprising an effective amount ofmiR-30c or a miR-30c agonist.
 10. A pharmaceutical composition for thetreatment of cardiovascular disease, hyperlipidemia, atherosclerosis,obesity, diabetes, or metabolic syndrome, comprising an effective amountof a miR-30c homolog.
 11. A pharmaceutical composition for treatingcardiovascular disease, hyperlipidemia, atherosclerosis, obesity,diabetes, or metabolic syndrome in a subject in need thereof, comprisingadministration of miR-30c or a miR-30c agonist in an amount effective totreat the disease in said subject wherein administration of the miR-30cor the miR-30c agonist results in avoidance of hepatic steatosis in saidsubject.
 12. A pharmaceutical composition for reducing serum lipids in asubject in need thereof, comprising administration of miR-30c or amiR-30c agonist in an amount effective to reduce serum lipids in saidsubject wherein administration of the miR-30c or the miR-30c agonistresults in avoidance of hepatic steatosis in said subject.
 13. Apharmaceutical composition for reducing microsomal triglyceride transferprotein (MTP) activity in a subject in need thereof, comprisingadministration of miR-30c or a miR-30c agonist in an amount effective toreduce MTP activity in said subject wherein administration of themiR-30c or the miR-30c agonist results in avoidance of hepatic steatosisin said subject.
 14. A pharmaceutical composition for reducing lipid orlipoprotein biosynthesis in a subject in need thereof, comprisingadministration of a miR-30c or a miR-30c agonist in an amount effectiveto reduce lipid or lipoprotein biosynthesis wherein administration ofthe miR-30c or the miR-30c agonist results in avoidance of hepaticsteatosis in said subject.
 15. The composition of claim 11, furthercomprising administration of at least one additional cholesterolreducing agent.
 16. The composition of claim 15, wherein one of the atleast one additional cholesterol reducing agents is a statin.
 17. Thecomposition of claim 12, further comprising administration of at leastone additional cholesterol reducing agent.
 18. The composition of claim1, wherein one of the at least one additional cholesterol reducingagents is a statin.
 19. The composition of claim 13, further comprisingadministration of at least one additional cholesterol reducing agent.20. The composition of claim 19, wherein one of the at least oneadditional cholesterol reducing agents is a statin.
 21. A pharmaceuticalcomposition for treating a disease or disorder selected from the groupconsisting of cardiovascular disease, hyperlipidemia, atherosclerosis,obesity, diabetes, or metabolic syndrome in a subject in need thereof,comprising administration of miR-30c or a miR-30c agonist in an amounteffective to reduce the levels of at least one target gene selected fromthe group consisting of LPGAT1, ELOVL5, MBOAT, IFG1R, ATP8B1, ALG9,LEPR, CHKA, PPARGC1A and StARD3 in said subject, wherein administrationof the miR-30c or the miR-30c agonist results in treatment of thedisease or disorder.
 22. The composition of claim 21, whereinadministration of the miR-30c or the miR-30c agonist results inavoidance of steatosis.
 23. A pharmaceutical composition for reducingserum lipids in a subject in need thereof, comprising administration ofmiR-30c or a miR-30c agonist in an amount effective to reduce the levelsof at least one target gene selected from the group consisting ofLPGAT1, ELOVL5, MBOAT, IFG 1 R, ATP8B 1, ALG9, LEPR, CHKA, PPARGC 1 Aand StARD3 in said subject, wherein administration of the miR-30c or themiR-30c agonist results in reducing serum lipids in said subject. 24.The composition of claim 23, wherein administration of miR-30c or themiR-30c agonist results in avoidance of steatosis.
 25. A pharmaceuticalcomposition for reducing microsomal triglyceride transfer protein (MTP)activity in a subject in need thereof, comprising administration of amiR-30c or a miR-30c agonist in an amount effective to reduce the levelsof at least one target gene selected from the group consisting ofLPGAT1, ELOVL5, MBOAT, IFG1R, ATP8B1, ALG9, LEPR, CHKA, PPARGC1A andStARD3 in said subject, wherein administration of the miR-30c or themiR-30c agonist results in reduced MTP activity in said subject.
 26. Thecomposition of claim 25, wherein administration of the miR-30c or amiR-30c agonist results in avoidance of steatosis.
 27. A pharmaceuticalcomposition for reducing lipid or lipoprotein biosynthesis in a subjectin need thereof, comprising administration of a miR-30c or a miR-30cagonist in an amount effective to reduce the levels of at least onetarget gene selected from the group consisting of LPGAT1, ELOVL5, MBOAT,IFG1R, ATP8B1, ALG9, LEPR, CHKA, PPARGC1A and StARD3 in said subject,wherein administration of the miR-30c or the miR-30c agonist results inreduced lipid or lipoprotein biosynthesis in said subject.
 28. Thecomposition of claim 27, wherein administration of the miR-30c or themiR-30c agonist results in avoidance of steatosis.